Progressive joint degeneration, as occurs in osteoarthritis, is common and current therapies for failing joints are limited. Attempts to induce repair of damaged articular cartilage in situ have had limited efficacy because this tissue seems to lack intrinsic regenerative capacity after birth. We hypothesize that understanding how pluripotent stem cells (PSCs) become articular chondrocytes (ACs) in vitro, and maintain this phenotype in vivo, will lead to improved strategies for repairing and regenerating cartilage. We will therefore identify genes and biological mechanisms responsible for enabling human (h)PSCs to become ACs. We will also identify factors that enable ACs to better resist becoming hypertrophic chondrocytes when challenged in vitro and implanted in vivo. Using our previously published methods for generating articular cartilage tissues from hPSCs, we will determine when these cells become fully committed to the AC lineage. We will accomplish this by inducing articular chondrocyte differentiation for different lengths of time using TGF?3, and then evaluating when the cells no longer become hypertrophic after BMP4 exposure in vitro and implantation into immunodeficient mice in vivo. In order to identify factors that maintain AC identity and enhance AC resilience in the presence these challenges, we will define the molecular profiles of hPSC-derived chondrocytes before and after they become ACs using RNA and ATAC sequencing. We will also compare the expression profiles of chondrogenic progenitors, articular chondrocytes, and growth plate chondrocytes derived from mouse ESC lines that we have CRISPR/Cas9 edited to express fluorescent proteins under the control of lineage-specific promoters. From these data, we will identify and prioritize for downstream studies candidate transcription factors, chromatin modifying enzymes, and signaling pathway components that are involved in AC lineage commitment. We will then assess the importance of individual candidates using genetic approaches (e.g., regulating gene expression using modified Cas9 proteins) and, when possible, pharmacologic approaches (e.g., pathway agonists and antagonists) in the hPSC/mESC differentiation assays. By understanding how ACs are induced from hPSCs and how the AC phenotype can be maintained in the presence of external challenges, we will discover new ways to repair, replace, or regenerate damaged cartilage in patients.